Early virologists called the time period between when infectious virus entered the host cell and when progeny virus was produced the eclipse period of infection because they could not readily determine what was going on using the techniques they had at hand. The experimental techniques outlined in this chapter have allowed modern virologists to visualize the eclipse period with the illumination of increasingly detailed knowledge. While the experimental analysis of virus infection takes time, money, and dedicated governmental interest, state of the art application of microrobotic techniques, laser-guided detection of target macromolecules interacting with substrates, and computer-enhanced quantitative measurement of such interactions, collectively termed microarray analysis, now provides the means of obtaining real-time measures of the intracellular environment as infection proceeds.
The basic idea behind microarray analysis is quite simple, and one example is illustrated in Fig. 12.13. In the most common versions, a large number of very small samples of individual target molecules, either nucleotide sequences complementary to cellular and viral genes or peptides known or thought to interact with host and virus-modified proteins, are bound to an inert substrate such as glass or a nylon membrane — the smaller the dimensions of the spots, the more samples that can be spotted on the matrix. Currently, sizes as small as 80 |m can be spotted, which means that a microscope slide can accommodate 10,000 or more different samples that are in use. This matrix containing the test material, with each variant spotted in a known location, is known as a microchip.
The microchip is then incubated with a small sample of a solution containing mixtures of macromolecules known or suspected to interact with the chip substrates. This could be mRNA or cDNA synthesized from mRNA if the chip contained fragments of DNA, or it could be a mixture of proteins from infected cells if the chip contained antibodies or peptides known to
Synthesize specific 75-mers
Robotically spot 4 ng aliquots in triplicate
Chemically activated glass slide
Oligonucleotide probe, thousands of spots 200 microns in diameter
Extract mRNA from infected cells mRNA
Make fluorescent labeled cDNA with oligo-dT or random octomer primers cDNA
Prepare slide for hybridization
Hybridize cDNA to oligonucleotide probe
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